Immunochemical and immunohistological expression of Lewis histo-blood group antigens in small intestine including individuals of the Le(a+b+) and Le(a−b−) nonsecretor phenotypes

Histological samples and total non-acid glycosphingolipids were prepared from small intestine of human cadavers with the Le(a+b+) and Le(a–b–) nonsecretor phenotypes and contrasted with the more common Lewis phenotypes. Glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with monoclonal antibodies reactive to Lewis epitopes. Paraffin-embedded small intestine sections were also fluorescently immunostained with anti-Lewis antibodies. Unlike the common Lewis positive phenotypes, we were immunochemically able to demonstrate the copresence of large amounts of Le^a and Le^b glycolipids in the Le(a+b+) sample. In addition we demonstrated increased formation of extended Lewis structures in this phenotype. By immunohistochemistry Le^a, Le^b and type 1 precursor chain epitopes could be demonstrated in the brush border. These results show that the expression of the Le(a+b+) phenotype at the erythrocyte phenotyping level parallels the small intestinal expression of this phenotype, and the patterns of Lewis antigen expressions are unique to this phenotype. By immunohistochemistry and immunochemistry we also demonstrated the presence of trace amounts of Lewis active glycoconjugates in the small intestine of the Le(a–b–) nonsecretor and Le(a+b–) samples. In the Le(a–b–) nonsecretor Le^a and Le^b activity was absent and type 1 precursor was present in brush border, while Le^b activity was immunohistologically demonstrated in the Golgi apparatus of the deep glands. Trace amounts of both Le^a and Le^b glycolipids were identified in this sample. In parallel trace Le^b activity could also be detected in the glycolipids of the Le(a+b–) sample and could be immunohistologically demonstrated to be fully expressed in occasional cells in the deep glands of the small intestine, a pattern quite dissimilar to that of the Le(a–b–) nonsecretor. The results in this paper show that the expression of Lewis glycoconjugates in the small intestine parallel the expression of Lewis erythrocyte phenotypes. However, inappropriate Lewis activity is also seen in individuals of other phenotypes and the mechanisms by which these Lewis antigens are made appears to be different for different phenotypes.Abbreviations FITC fluorescein isothiocyanate - HPLC high-performance liquid chromatography - NeuAc N-acetyl-d-neuraminic acid - RBC red blood cell - TLC thin-layer chromatography - TRITC tetramethyl rhodamine isothiocyanate     

Expression of Lewis histo-blood group glycolipids in the plasma of individuals of Le(a+b+) and partial secretor phenotypes

Red cell Lewis antigens are carried by glycosphingolipids passively absorbed from plasma. Plasma was collected from a spectrum of individuals with normal and unusual Lewis/secretor phenotypes in order to investigate the glycolipid basis for the unusual phenotypes. Samples were obtained from: a Le(a+b–) ABH nonsecretor who secreted Lewis substances; a Le(a+b–) partial secretor; Le(a+b+) partial secretors; Le(a+b+) secretors; and a full range of normal Lewis/secretor phenotypes as controls. The Le(a+b+) samples represented Polynesian, Asian and Réunion Island ethnic backgrounds. Nonacid glycolipids were prepared, separated by thin-layer chromatography, and then immunostained with potent monoclonal antibodies of known specificity. Despite different serological profiles of the Le(a+b–) and Le(a+b+) Polynesian samples, their plasma glycolipid expressions were very similar, with both Le^a and Le^b co-expressed. The copresence of Le^a and Le^b in Le(a+b+) samples is in marked contrast to Caucasians with normal Lewis phenotypes, who have predominantly either Le^a or Le^b. These results suggest that there is a range of the secretor transferases in different individuals, possibly due to different penetrance or to several weak variants. We also show that Lewis epitopes on longer and/or more complex core chains appear to be predominant in the Polynesian Le(a+b+) samples. The formation of these extended glycolipids is compatible with the concept that in the presence of reduced secretor fucosyltransferase activity, increased elongation of the precursor chain occurs, which supports the postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.Abbreviations CBA chromatogram binding assay - TLC thin-layer chromatography     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

A Common Region of Deletion on Chromosome 17q in Both Sporadic and Familial Epithelial Ovarian Tumors Distal to BRCA1

Linkage analysis in familial breast and ovarian cancer and studies of allelic deletion in sporadic ovarian tumors have identified a region on chromosome 17q containing a candidate tumor-suppressor gene (referred to as BRCA1) of likely importance in ovarian carcinogenesis. We have examined normal and tumor DNA samples from 32 patients with sporadic and 8 patients with familial forms of the disease, for loss of heterozygosity (LOH) at 21 loci on chromosome 17 (7 on 17p and 14 on 17q). LOH on 17p was 55% (22/40) for informative 17pl3.1 and 17pl3.3 markers. When six polymorphic markers flanking the familial breast/ovarian cancer susceptibility locus on 17ql2-q21 were used, LOH was 58% (23/40), with one tumor showing telomeric retention. Evaluation of a set of markers positioned telomeric to BRCA1 resulted in the highest degree of LOH, 73% (29/40), indicating that a candidate locus involved in ovarian cancer may reside distal to BRCA1. Five of the tumors demonstrating allelic loss for 17q markers were from individuals with a strong family history of breast and ovarian cancer. More important, two of these tumors (unique patient number [UPN] 57 and UPN 79) retained heterozygosity for all informative markers spanning the BRCA1 locus but showed LOH at loci distal to but not including the anonymous markers CMM86 (D17S74) and 42D6 (D17S588), respectively. Deletion mapping of seven cases (two familial and five sporadic) showing limited LOH on 17q revealed a common region of deletion, distal to GH and proximal to D17S4, that spans −25 cM. These results suggest that a potential tumor-suppressor gene involved in both sporadic and familial ovarian cancer may reside on the distal portion of chromosome 17q and is distinct from the BRCA1 gene.     

Use of Genetic and Physical Mapping to Locate the Spinal Muscular Atrophy Locus between Two New Highly Polymorphic DNA Markers

The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5ql3, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112. We identified two new highly polymorphic microsatellite DNA markers—namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)—which are the closest markers to the SMA locus. Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357. Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen–D5S6–D5S125/D5S465–D5S435–D5S629–SMA–D5S637–D5S351–MAP–1B/D5S112–D5S357–D5S39–tel. These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.     

Simulation Model of the Coupling between Nitrification and Denitrification in a Freshwater Sediment

A model was constructed to simulate the results of experiments which investigated nitrification and denitrification in the freshwater sediment of Lake Vilhelmsborg, Denmark (K. Jensen, N. P. Sloth, N. Risgaard-Petersen, S. Rysgaard, and N. P. Revsbech, Appl. Environ. Microbiol. 60:2094-2100, 1994). The model output faithfully represented the profiles of O₂ and NO₃^- and rates of nitrification, denitrification, and O₂ consumption as the O₂ concentration in the overlying water was increased from 10 to 600 μM. The model also accurately predicted the response, to increasing O₂ concentrations, of the integrated (micromoles per square meter per hour) rates of nitrification and denitrification. The simulated rates of denitrification of NO₃^- diffusing from the overlying water (D_w) and of NO₃^- generated by nitrification within the sediment (D_n) corresponded to the experimental rates as the O₂ concentration in the overlying water was altered. The predicted D_w and D_n rates, as NO₃^- concentration in the overlying water was changed, closely resembled those determined experimentally. The model was composed of 41 layers 0.1 mm thick, of which 3 represented the diffusive boundary layer in the water. Large first-order rate constants for nitrification and denitrification were required to completely oxidize all NH₄⁺ diffusing from the lower sediment layers and to remove much of the NO₃^- produced. In addition to the flux of NH₄⁺ from below, the model required a flux of an electron donor, possibly methane. Close coupling between nitrification and denitrification, achieved by allowing denitrification to tolerate some O₂ (~10 μM), was necessary to reproduce the real data. Spatial separation of the two processes (no toleration by denitrification of O₂) resulted in too high NO₃^- concentrations and too low rates of denitrification.     

Co-localization of cytokinins with proteins related to cell proliferation in developing somatic embryos of Dactylis glomerata L.

The present report provides evidence for co-localization ofcytokinins with cell proliferation-associated nuclear proteins.Somatic embryos of Dactylis glomerata in two stages of developmentare used as a model system comprising both proliferating andinitially differentiated cells. Cytokinins are localized usingantibodies with marked specificity against isopentenyladenine/adenosine(2iP/2iPA) or zeatin/ riboside (Z/ZR). The proliferation-associatednuclear antigen, mitotin, is analysed using a specific monoclonalantibody. The nuclear protein BM28, required for the onset ofDNA replication and for cell division, is identified by an affinity-purifiedpolyclonal antibody. Using double immunofiuorescence labellingwith the antibodies against cytokinins and against each of thenuclear proteins, immunoreaction is observed generally in thesame nuclei of almost all cells in globular embryos and in thenuclei of cells in meristematic areas of the more developedembryos. Only small numbers of individual nuclei positive forboth type of antibodies were found in the surrounding vacuolatedparenchymatous cells. The occurrence of plant antigens homologousto BM28 and mitotin is confirmed by immunoblotting assay. InSDS-PAGE blots the anti-BM28 antibody reacts with a proteinof 58 kDa. The anti-mitotin antibody recognizes several (160,140, 125, 93, and 80 kDa) polypeptides. The data showing nuclearco-localization of cytokinins and proteins with a suggestedrole in the onset of DNA synthesis and in cell division providea new base for further study on the mode of action of cytokininsin cell cycle regulation. Key words: Immunolocalization, cytokinins, nuclear proteins, mitotin, BM28, cell proliferation, somatic embryo(s), Dactylis glomerata     

A Pleiotropic Phospholipid Biosynthetic Regulatory Mutation in Saccharomyces Cerevisiae Is Allelic to Sin3 (Sdi1, Ume4, Rpd1)

Three mutants were identified in a genetic screen using an INO1-lacZ fusion to detect altered INO1 regulation in Saccharomyces cerevisiae. These strains harbor mutations that render the cell unable to fully repress expression of INO1, the structural gene for inositol-1-phosphate synthase. The Cpe(-) (constitutive phospholipid gene expression) phenotype associated with these mutations segregated 2:2, indicating that it was the result of a single gene mutation. The mutations were shown to be recessive and allelic. A strain carrying the tightest of the three alleles was examined in detail and was found to express the set of co-regulated phospholipid structural genes (INO1, CHO1, CHO2 and OPI3) constitutively. The Cpe(-) mutants also exhibited a pleiotropic defect in sporulation. The mutations were mapped to the right arm of chromosome XV, close to the centromere, where it was discovered that they were allelic to the previously identified regulatory mutation sin3 (sdi1, ume4, rpd1, gam2). A sin3 null mutation failed to complement the mutation conferring the Cpe(-) phenotype. A mutant harboring a sin3 null allele exhibited the same altered INO1 expression pattern observed in strains carrying the Cpe(-) mutations isolated in this study.     

Sheep Linkage Mapping: Nineteen Linkage Groups Derived from the Analysis of Paternal Half-Sib Families

Nineteen linkage groups containing a total of 52 markers have been identified in the sheep genome after typing large paternal half-sib families. The linkage groups range in size from 2 markers showing no recombination to a group containing 6 markers covering approximately 30 cM of the sheep genome. Thirteen of the groups have been assigned to a sheep chromosome. Three groups contain markers from bovine syntenic groups U2, U7 and U29, and one other group contains a marker that has been mapped only in humans. The remaining three groups are unassigned. This information will provide a useful foundation for a genetic linkage map of sheep.